High-pressure liquid chromatography (HPLC) is a process used for separating one or more compounds from a chemical mixture. The HPLC process consists of passing the mixture through a stationary packing material, under the influence of a high-pressure transport liquid, and separating the compounds by selective affinity, sieving, adsorption or partitioning. The packing is typically housed within a chamber formed in a cylindrical column and is typically held in place by frits at either end of the chamber. A guard column, either as a distinct mechanism or as a column butted against the HPLC column, can be used to protect the HPLC column from physical or chemical contamination.
The guard column traps impurities or particulates in the sample mixture or in the transport fluid before the impurities or particulates can reach the HPLC column and thereby extends the operable life of the HPLC column. It is desirable to be able to remove a guard column. The advantage of the replaceable guard column is that the HPLC column, which is substantially more expensive than the guard column, does not have to be replaced as often. One concern with guard columns in HPLC equipment is assuring that the connection between the guard column and the HPLC column is sealable and removable. At the high pressures used, simple connection and sealing mechanisms have not sufficed. Therefore, the attachment mechanism increases the overall cost of the column.
As HPLC is performed on more minute quantities of samples, the size of the columns is reduced. Nanocolumns of 75 μm ID are commonly used for small samples. The use of 75 μm ID nanocolumns is often a challenge to the user who needs to analyze complex or “dirty” samples. The nanocolumns loaded with such samples frequently block after one or two injections and are rendered useless for further analysis. Due to the small scale of the nanocolumns, any guard column used with the nanocolumns would have to be installed with virtually no dead space in order to avoid bandspreading. Such guard columns have not been available. In addition, any joining mechanism for attaching a guard column to a nanocolumn must withstand high pressures even though the joining mechanism needs to be very small.
In another aspect of HPLC, there are occasions when it is advantageous to create a column having two distinct analytical regions. These regions must be precisely formed as to composition and length and separated by a neutral material, typically a frit, in order to accomplish a specific separation. As the inside diameter of the cylinder forming the column is reduced, the ability to form separate analytical regions becomes compromised. In nanocolumns, it has not been possible to separate the regions. Consequently, such multi-use nanocolumns are not commercially available. In a research setting, the only multi-use columns that have been fabricated do not have a definite demarcation between the analytical regions. There is an area that is a mixture of the two media at the junction that renders these multi-use columns irreproducible.
There is currently a need to extend the life of nanocolumns even when they are used to analyze complex samples.